Microbial production of actinol

ABSTRACT

A process for making (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone by contacting (6R)-2,2,6-trimethylcyclohexanedione with a microorganism which is selected from microorganisms of the genera Cellulomonas, Corynebacterium, Planococcus and Arthrobacter and which is capable of the selective asymmetric reduction of (6R)-2,2,6-trimethylcyclohexanedione to (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, and recovering the resulting (4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone from the reaction mixture. The selective asymmetric reduction can be effected in the presence of a co-factor, such as, nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), or said co-factor with glucose and glucose dehydrogenase (GDH), and/or in the presence of a surfactant. The product is useful for the synthesis of carotenoids, such as, zeaxanthin.

FIELD OF THE INVENTION

The present invention relates to a process for the microbial productionof (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone (“actinol”) from(6R)-2,2,6-trimethylcyclohexanedione (“levodione”). Actinol is usefulfor the synthesis of carotenoids, such as zeaxanthin. More particularly,the present invention relates to a process for the microbial productionof actinol utilizing a specific microorganism that is capable ofselectively asymmetrically reducing the carbonyl group at the C-4position of levodione.

BACKGROUND OF THE INVENTION

Actinol has previously been prepared by optical resolution of thediastereomeric mixture of actinol. However, this process requireshydrogenation of levodione by metal catalysts and subsequent opticalresolution by chemical means with resolving agents, such as, maleicanhydride (T. Ohashi et al., the proceedings of the symposium “MolecularChirality 1996” held on May 30 and 31, 1996, in Tokyo, Japan, pages 47to 50, “Practical Syntheses using Biocatalysts”). Accordingly, thisprocess is not economically feasible for industrial purposes.

Processes of enzymatic preparation of actinol from levodione per se areknown. For example, Bacillus thermophilus is capable of convertingracemic dihydrooxoisophorone to 4 isomers of4-hydroxy-2,2,6-trimethylcyclohexanone, i.e., to the cis-(4R,6S)-,cis-(4S,6R)-, trans-(4R,6R)- and trans-(4S,6S)-isomers. The resultingquantitative ratio of these isomers is 68:25:5:2 (J. Biotechnol., 9(2),117 -128, 1989). Because the content of the (4R,6R)-isomer, actinol, isonly 5% of the total isomers, this process is also not economicallyfeasible for industrial purposes.

SUMMARY OF THE INVENTION

As a result of extensive studies on selective asymmetric reduction oflevodione, it has surprisingly been found that actinol can be obtainedefficiently from levodione by selective asymmetric reduction usingcertain microorganisms followed by recovery of the actinol from thereaction mixture. The present invention is based upon this finding.

Accordingly, the present invention provides a process for making actinolby contacting levodione with a microorganism, which is selected from thegroup consisting of microorganisms of the genera Cellulomonas,Corynebacterium, Planococcus and Arthrobacter and which is capable ofselective asymmetric reduction of levodione to actinol, and recoveringthe resulting actinol from the reaction mixture.

DETAILED DESCRIPTION OF THE INVENTION

Screening was effected using a method known per se. For example, amicroorganism is cultivated in a nutrient medium containing saccharides,such as, glucose and sucrose, alcohols, such as, ethanol and glycerol,fatty acids, such as, oleic acid and stearic acid or esters thereof, oroils, such as, rapeseed oil and soybean oil as carbon sources; ammoniumsulfate, sodium nitrate, peptone, amino acids, corn steep liquor, bran,yeast extract and the like as nitrogen sources; magnesium sulfate,sodium chloride, calcium carbonate, potassium monohydrogen phosphate,potassium dihydrogen phosphate and the like as inorganic salt sources;and malt extract, meat extract and the like as other nutrient sources bya conventional method to provide cells. The cultivation can be carriedout aerobically, normally for a cultivation period of 1 to 7 days at amedium pH of 3 to 9 and a cultivation temperature of 10 to 40° C. Afterthe cultivation, the resulting cells are collected by centrifugation orfiltration. The cells thus obtained and levodione are brought(contacted) together in a solvent such as water, potassium phosphatebuffer, acetonitrile, ethanol and the like, and a reaction is initiatedunder appropriate reaction conditions (levodion concentration: 400 to2000 mg/g dry cells/l, pH range: 4 to 9, temperature range: 20 to 50°C., reaction period: 10 minutes to 80 hours). The reaction mixture isextracted with an organic solvent such as ethyl acetate, n-hexane,toluene, n-butyl acetate and the like. The extracted solution issubjected to an appropriate method, such as, chromatography, to measurethe productivity of actinol from levodione.

As a result of the screening, it has been found that microorganismsbelonging to the genera Cellulomonas, Corynebacterium, Planococcus andArthrobacter are capable of the selective asymmetric reduction oflevodione. Preferred such microorganisms are Cellulomonas sp. AKU672,Corynebacterium aquaticum AKU610, Corynebacterium aquaticum AKU611,Planococcus okeanokoites AKU152 and Arthrobacter sulfurous AKU635.Especially preferred are the first three named microorganisms, of whichCorynebacterium aquaticum AKU611 is most preferred.

The microorganisms Cellulomonas sp. AKU672, Corynebacterium aquaticumAKU610 and Corynebacterium aquaticum AKU611 were isolated from soilsamples collected at Lake Manahime, Fukui Prefecture, Japan. Thesemicoorganisms were deposited with the National Institute of Bioscienceand Human-Technology, Agency of Industrial Science and Technology, Japanon Aug. 4, 1998 under the Budapest Treaty and have the followingdesignations: Cellulomonas sp. AKU672 (FERM BP-6449) Corynebacteriumaquaticum AKU610 (FERM BP-6447) Corynebacterium aquaticum AKU611 (FERMBP-6448)

Cellulomonas sp. AKU672, Corynebacterium aquaticum AKU610, andCorynebacterium aquaticum AKU611 were deposited on Aug. 4, 1998 at theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology (1-3, Higashi 1-Chome, Tsukuba City,Ibaraki Pref., Japan) as FERM BP-6449, FERM BP-6447, FERM BP-6448,respectively under the terms of the Budapest Treaty. All restrictions onpublic access to these deposits will be irrevocably removed upon thegrant of a patent on this application.

These three microorganisms, and also Planococcus okeanokoites AKU152 andArthrobacter sulfureus AKU635, are new and represent a further aspect ofthe present invention.

The above-mentioned strain AKU672 (FERM BP-6449) has the followingtaxonomical properties:

Typical pleomorphism of strain Cellulomonas sp. AKU672 was found onelectron microscopic observation. An old culture of the strain wascoccoidal as shown in FIG. 1. In young cultures, irregular rods weredominant (FIG. 2). The morphological, physiological and biochemicalcharacteristics of the strain are summarized in Tables I and II.

TABLE I Morphological and Culture Characteristics of Strain Cellulomonassp. AKU672 Form and size Coryneform Old culture; coccoid cells, ca.0.5-0.6 μm Fresh culture; irregular rods, 0.5-0.7 μm by 20 or more μmMotility Motile with one flagellum Gram strain + Spores No observationNutrient agar plate Circular, convex, smooth, entire, yellow (2 days)Nutrient broth Ring and slight sediment Gelatin stab Liquefaction Litmusmilk Acid formation Relation to NaCl Grow up to 5% NaCl

TABLE II Physiological and Biochemical Characteristics of StrainCellulomonas sp. AKU672 Type of cell wall Ornithine Type of celldivision Bending GC content (%) 74.7% Hydrolysis of gelatin + Hydrolysisof starch + Production of indole − Production of hydrogen − sulfideReduction of nitrate + to nitrite Utilization of citrate − Catalaseactivity + Oxidase activity − Urease activity − DNase activity + Aminopeptidase − activity Cellulose attack − Voges-Proskauer test −Methyl-red test − Oxidation- Fermentation Fermentation test* Cleavage ofAcid production but no gas from arabinose, carbohydrates arbutin,cellobiose, dextrin, fructose, galactose, glucose, glycogen, maltose,starch, sucrose, trehalose and xylose; No acid production from glycerol,inulin, lactose, mannitol, mannose, α- methylglucoside, raffinose,rhamnose, sorbitol and sorbose Optimum temperature 37-42° C. for growthOptimum pH for pH 6.0-7.5 growth Heating at 63° C. for Survives 30 min.in skimmed milk Aerobic or anaerobic Aerobic *R. Hugh & E. Leifson, J.Bacteriol. 66, 24(1953)

The strain Cellulomonas sp. AKU672 is gram-positive and aerobic, and canbe classified as belonging to the group of “coryneform bacteria”. Thisstrain was motile with one flagellum. Ornithine was found in the cellwall as the principal amino acid. Its content according to gaschromatography (“GC”) was found to be 74.7%. Bending-like cell divisionwas observed. The strain produced acid from a wide variety of sugarswithout gas formation for 4 days. This strain did not show cellulolyticactivity.

The classification of coryneform bacteria is not well established.Recently, Yamada and Komagata [J. Gen. Appl. Microbiol., 18, 417 (1992)]proposed classifying the coryneform bacteria into seven groups dependingon the principal type of cell division, cell wall composition and DNAcontent according to GC. They differentiated Group 4 from other groupsdespite the lack of cellulolytic activity. Bacteria of this groupexhibit the bending type of cell division, and the principal amino acidin the cell wall is ornithine. Their contents according to GC aredistributed in a narrow and high range from 71 to 73%. These bacteriaproduce acid fermentatively from a wide variety of sugars. According totheir proposal, the strain Cellulomonas sp. AKU672, which did not showcellulolytic activity, should belong to Group 4. Other characteristicsof the strain on classification coincided well with those of Group 4,and so it has been tentatively named as Cellulomonas sp. AKU672.

The above-mentioned strains AKU610 and AKU611 have the followingtaxonomical properties:

1) Growable temperature: 15-40° C.

2) Optimum temperature for growth: 30° C.

3) Obligatory aerobic and gram negative microorganism

4) Spore formation: None

5) Polymorphism and traditional rod-cocus cycles can be observed duringcultivation.

6) Motility: None

Moreover, the strains Corynebacterium aquaticum AKU610 and AKU611 wereidentified as such based on assimilation of various carbon sources bythe Biolog System (Biolog Inc., 3447 Investment Blvd., Suite 3, Hayward,Calif. 94545, USA: Nature Vol. 339, 157-158, May 11, 1989) as follows:

Cells of each strain were inoculated with 96-well microtiter-plates andincubated for 24 hours at 28° C. Each well contains one of 96 kinds ofcarbon sources in BUGM+B medium (Biolog Universal Growth Media+blood;Biolog Inc.).

After incubation, each strain showed the following assimilation ofcarbon sources:

C source AKU610 AKU611 C source AKU610 AKU611 A1 − − A2 − − A3 − − A4 −− A5 − − A6 − − A7 − − A8 + + A9 + + A10 − − A11 − − A12 + + B1 − − B2− + B3 − − B4 + − B5 + + B6 − − B7 + + B8 − − B9 + + B10 + + B11 + + B12− − C1 − − C2 − − C3 − − C4 + + C5 + + C6 + + C7 + − C8 + + C9 − − C10 −− C11 − − C12 − − D1 − − D2 − − D3 + + D4 − − D5 + + D6 − − D7 − − D8− + D9 − − D10 − − D11 + + D12 + + E1 − − E2 − − E3 + + E4 − − E5 − − E6− − E7 − − E8 − − E9 − − E10 − − E11 − − E12 − − F1 − − F2 − − F3 − − F4− − F5 − − F6 + + F7 − − F8 − − F9 − − F10 − − F11 − − F12 − − G1 − − G2− − G3 − − G4 − − G5 − − G6 − − G7 − − G8 − − G9 − − G10 − − G11 − − G12− − H1 − − H2 − − H3 − − H4 − − H5 − − H6 − − H7 − − H8 − − H9 − − H10 −− H11 − − H12 − − A1: water A2: α-cyclodextrin A3: β-cyclodextrin A4:dextrin A5: glycogen A6: inulin A7: mannan A8: Tween ® 40 A9: Tween ® 80A10: N-acetyl-D-glucosamine A11: N-acetyl-D-mannosamine A12: amygdalinB1: L-arabinose B2: D-arabitol B3: arbutin B4: cellobiose B5: D-fructoseB6: L-fucose B7: D-galactose B8: D-galacturonic acid B9: gentiobioseB10: D-gluconic acid B11: α-D-glucose B12: m-inositol C1: α-D-lactoseC2: lactulose C3: maltose C4: maltotriitrose C5: D-mannitol C6:D-mannose C7: D-melezitose C8: D-melibiose C9: α-methyl-D-galactosideC10: α-methyl-D-galactoside C11: 3-methyl-glucose C12:α-methyl-D-glucoside D1: β-methyl D-glucoside D2: α-methyl D-mannosideD3: palatinose D4: D-psicose D5 D-raffinose D6: L-rhamnose D7: D-riboseD8: salicin D9: sedoheputulosan D10: D-sorbit D11: stachyose D12:sucrose E1: D-tagatose E2: D-trehalose E3: turanose E4: xylitol E5:D-xylose E6: acetic acid E7: α-hydroxybutyric acid E8: β-hydroxybutyricacid E9: γ-hydroxybutyric acid E10: p-hydroxyphenylacetic acid E11:α-keto-glutaric acid E12: α-keto-valeric acid F1: lactamide F2: D-lacticacid methyl ester F3: L-lactic acid F4: D-malic acid F5: L-malic acidF6: methyl pyruvate F7: monomethyl succinate F8: propionic acid F9:pyruvic acid F10: succinamic acid F11: succinic acid F12:N-acetyl-L-glutamic acid G1: alaninamide G2: D-alanine G3: L-alanine G4:L-alanyl-glycine G5: L-asparagine G6: L-glutamic acid G7:glycyl-L-glutamic acid G8: L-pyloglutamic acid G9: L-serine G10:putrscine G11: 2,3-butanediol G12: glycerol H1: adenosine H2: 2′-deoxyadenosine H3: inosine H4: thymidine H5: uridine H6:adenosine-5′-monophosphate H7: thymidine-5′-monophosphate H8:uridine-5′-monophosphate H9: fructose-6-phosphate H10:glucose-1-phosphate H11: glucose-6-phosphate H12: DL-α-glycerolphosphate

From the above results, both strains are identified as Corynebacteriumaquaticum and named Corynebacterium aquaticum AKU610 and AKU611,respectively.

Other microorganisms mentioned above are available from a publicdepositary (culture collection) to anyone upon request, such as theInstitute of Fermentation Osaka, Japan (IFO). Examples of such depositedstrains are Planococcus okeanokoites AKU152 (IFO 15880) and Arthrobactersulfureus AKU635 (IFO 12678).

The selective asymmetric reduction process of the present invention canbe carried out batchwise, semibatchwise or continuously in water or in asolvent medium that is miscible with water, enhances levodionesolubility and is inert to the enzyme reaction, such as, 0.01 to 0.5Mpotassium phosphate buffer, another buffer with the pH range 4 to 10,acetonitrile, ethanol or N,N-dimethylformamide. The concentration oflevodione is conveniently 400 to 2000 mg/1 g dry cells/l, preferably 400to 800 mg/1 g dry cells/l. The selective asymmetric reduction processmay be carried out in a pH range from 4 to 9, preferably from 6 to 7, ina temperature range from 20 to 50° C., preferably 30 to 40° C., and for10 minutes to 80 hours, preferably for 8 hours to 24 hours.

The selective asymmetric reduction process of the present invention isconveniently carried out in the presence of a co-factor such asnicotinamide adenine dinucleotide (NAD), nicotinamide adeninedinucleotide phosphate (NADP), or said co-factor with glucose andglucose dehydrogenase (GDH). The concentration of such co-factor in thereaction medium is preferably 300 mM/l or more, more preferably from 700mM/l to 900 mM/l. Moreover, the yield of actinol can be increased byaddition of a surfactant to the reaction mixture. Span® 20, Span® 80,Tween® 20, Tween® 40 (all available from Wako Pure Chemical Ind., 3-1-2Dosho-machi, Osaka, Japan) and the like are examples of surfactants thatcan be used. The amount of surfactant in the reaction medium isconveniently 2 to 20 mM/l, preferably about 8 mM/l.

After selective asymmetric reduction has been completed, the actinolthus obtained can be recovered by extraction with a water-insoluble(water- immiscible) organic solvent that readily solubilizes actinol,such as, ethyl acetate, n-hexane, toluene or n-butyl acetate. Furtherpurification of actinol can be effected by concentrating the extract todirectly crystallize the actinol or by the combination of various kindsof chromatography, such as, thin layer chromatography, adsorptionchromatography, ion-exchange chromatography and/or gel filtrationchromatography. If necessary, high performance liquid chromatography canalso be applied. A preferred recovery leading to crystals of actinolinvolves extracting the actinol with ethyl acetate and concentrating theextract to obtain actinol crystals.

As an alternative to the above described “resting cell reaction”technique, actinol can be produced by fermentation of the abovemicroorganisms in a nutrient medium in the presence of levodione, i.e.,in a “growing cell reaction”. Both alternatives are embraced by theprocess of the present invention.

As nutrient media in the “growing cell reaction” technique there may beused those which contain saccharides, such as, glucose and sucrose,alcohols, such as, ethanol and glycerol, fatty acids, such as, oleicacid and stearic acid or esters thereof, or oils, such as, rapeseed oiland soybean oil as carbon sources; ammonium sulfate, sodium nitrate,peptone, amino acids, corn steep liquor, bran, yeast extract and thelike as nitrogen sources; magnesium sulfate, sodium chloride, calciumcarbonate, potassium monohydrogen phosphate, potassium dihydrogenphosphate and the like as inorganic salts; and malt extract, meatextract and the like as other nutrient sources. As a further aspect ofthe present invention, actinol can be produced by fermentation of theabove microorganisms in a nutrient medium in the presence of levodione.

The fermentation can be carried out aerobically, normally for anincubation period of 1 to 7 days at a medium pH of 3 to 9 and afermentation temperature of 10 to 40° C.

The microorganisms to be used in the fermentation may be in any form,for example, cultures obtained by fermentation of strains in liquidmedia, cells separated from liquid cultures, dried cells obtained byprocessing cells or cultures, or immobilized cells.

The following Examples illustrate the present invention.

EXAMPLE 1

A liquid medium (pH 7.0) having 0.5% 1,4-cyclohexanedione (structurallyanalogous to (6R)-2,2,6-trimethylcyclohexanedione; used for thescreening), 0.5% Tween® 20, 0.1% (NH₄)₂SO₄, 0.1% K₂HPO₄, 0.02%MgSO₄.7H₂O and 0.02% yeast extract was dispersed in 5 ml portions intotest tubes, and then sterilized at 121° C. for 20 minutes. About 0.3 gof soil sample was introduced into each of these tubes and cultivatedfor 24 hours at 30° C. A 0.1 ml portion of the culture thus obtained wasused to inoculate fresh test tube medium as above, and this operationwas repeated twice. The enriched culture thus obtained was diluted withsaline and spread on an agar medium having the same ingredients asabove. Simultaneously, supernatant of the soil suspension in saline wasappropriately diluted and spread on the agar medium as well. The plateswere incubated for 48 hours at 30° C. Grown colonies on the plates wereused to inoculate 5 ml liquid medium (pH 7.0) having 1.0% glucose, 0.3%K₂HPO₄, 0.02% MgSO₄.7H₂O, 1.5% peptone (Mikuni Kagaku Sangyo K. K.,4-1-6 Muro-machi, Nihonbashi, Chuo-ku,Tokyo, Japan), 0.2% NaCl and 0.1%yeast extract (Nacalai Tesuque Inc., Karasumaru Nishihairu, Nijohtouri,Nakakyo-ku, Kyoto, Japan) in a tube. After the tubes had been incubatedat 30° C. for 24 hours, cells were collected by centrifugation andwashed with saline. The cells thus obtained were subjected for thesubsequent screening. In addition to the above microorganisms, air-driedcells of the microorganisms that had been cultivated in a nutrientmedium were also used for the screening.

EXAMPLE 2

A reaction mixture (pH 7.0 in 0.1 M potassium phosphate buffer)containing 0.6 mg of NAD (Oriental Yeast Co., 3-6-10 Azusawa,Itabashi-ku, Tokyo, Japan), 0.6 mg of NADP (Oriental Yeast Co.), 50 mgof D-glucose and 0.2 mg of D-glucose dehydrogenase (Amano PharmaceuticalCo., 1-2-7 Nishiki, Naka-ku, Nagoya, Japan) was prepared. About 0.3 g ofthe cells prepared in Example 1 was added to 1 ml of the reactionmixture, followed by a sufficient amount of(6R)-2,2,6-trimethylcyclohexanedione to give a final concentration of0.5%. The reaction mixture was then incubated with shaking for 24 hoursat 30° C. After incubation, the reaction mixture was extracted with 1 mlof ethyl acetate and concentrated. The yield and the optical purity ofthe (4R, 6R)-4-hydroxy-2,2,6-trimethyl-cyclohexanone were analyzed bygas chromatography [column: HR-20M (Shinwa Chemical Ind., Keishyo-cho50, Fushimi-ku, Kyoto, Japan) 0.25 mmφ×30 m, column temperature: 160° C.(constant), injector temperature: 250° C., carrier gas: He (approx. 1ml/min)]. The results are presented in Table III.

TABLE III Rate of Optical purity of (4R, 6R)- reduction4-hydroxy-2,2,6-trimethyl- Strain Name (%) cyclohexanone (% e.e.)Planococcus okeanokoites 42.3 56.7 AKU152 (IFO 15880) Arthrobactersulfureus 64 44 AKU635 (IFO 12678) Cellulomonas sp. AKU672 73 78.3 (FERMBP-6449) Corynebacterium aquaticum 93.7 85.9 AKU610 (FERM BP-6447)Corynebacterium aquaticum 97.4 87.7 AKU611 (FERM BP-6448)

EXAMPLE 3

The effect of the addition of NAD or NADP to the reaction mixture waselucidated by using the microorganisms given in Table III. The basicreaction mixture contained all the components described in Example 2except NAD and NADP. The cells of the microorganisms used in the presentExample were air-dried, and 10 mg of the cell mass were incorporatedinto the reaction mixture. The reaction was carried out at 30° C. for 24hours. The results are presented in Table IV, in which the opticalpurity (% e.e.) values apply to the (4R,6R)-isomer, as is also the casein Tables V (Example 4) and VI (Example 5).

TABLE IV Co-factor Addition NAD NADP None Rate of Optical Rate ofOptical Rate of Optical reduction purity reduction purity reductionpurity Strain Name (%) (% e.e.) (%) (% e.e.) (%) (% e.e.) Planococcus89.3 60.4 65.4 54.7 63.4 58.2 okeanokoites AKU152 (IFO 15880)Arthrobacter 82.7 24   66.5 −7.3 56.5 −9.5 sulfureus AKU635 (IFO 12678)Cellulomonas sp. 59.2 67.1 30.1 21.6 24.8 25.7 AKU672 (FERM BP-6449)Corynebacterium 62.5 87.4 60   85.3 17   52.1 aquaticum AKU610 (FERMBP-6447) Corynebacterium 96.8 93.9 85.3 88.5 92.5 89.1 aquaticum AKU611(FERM BP-6448)

EXAMPLE 4

The effect of the addition of various surfactants (final concentration:0.1 w/v %) in the reaction mixture was elucidated by using themicroorganisms given in Table III. The basic reaction mixture containedall the components described in Example 2. The cells of themicroorganisms used in the present Example were air-dried, and 10 mg ofthe cell mass were incorporated into the reaction mixture. The reactionwas carried out at 30° C. for 24 hours. The results are presented inTable V.

TABLE V Surfactant None Tween ® 20 Tween ® 40 Span ® 20 Span ® 80 Rateof Optical Rate of Optical Rate of Optical Rate of Optical Rate ofOptical reduction purity reduction purity reduction purity reductionpurity reduction purity Strain Name (%) (% e.e.) (%) (% e.e.) (%) (%e.e.) (%) (% e.e.) (%) (% e.e.) Planococcus 53.9 51.6 78.1 63.4 63.957.7 71.4 65.7 57.3 57.3 okeanokoites AKU152 (IFO 15880) Arthrobacter73.4 25.1 86.8 38.6 82.4 33.8 78.5 49.9 65.2 32   sulfureus AKU635 (IFO12678) Cellulomonas sp. 32.3 80   32.1 86.2 not n.m. 22.2 66.2 38   78.1AKU672 measured (FERM BP-6449) (n.m.) Corynebacterium 58.9 87.6 71.789.3 n.m. n.m. 64.6 89.9 83   87.3 aquaticum AKU610 (FERM BP-6447)Corynebacterium 85.7 92.6 97.5 93.7 n.m. n.m. 96.7 94   88.8 93.2aquaticum AKU611 (FERM BP-6448)

EXAMPLE 5

The influence of the substrate concentration on the reaction waselucidated at concentrations of 0.5, 1.0 and 1.5%. The basic reactionmixture contained all the components described in Example 2. In thepresent Example, the cells of Corynebacterium aquaticum AKU611 (FERMBP-6448) were air-dried, and 10 mg of the cell mass were incorporatedinto the reaction mixture. The reaction was carried out at 30° C. for 24hours. The results are represented in Table VI.

TABLE VI Rate of Substrate reduction Optical purity ProductConcentration (%) (%) (% e.e.) concentration (%) 0.5 92.2 93.0 0.46 1.073.1 92.9 0.73 1.5 66.3 92.8 0.99

EXAMPLE 6

Corynebacterium aquaticum AKU611 (FERM BP-6448) was cultivated for 24hours at 30° C. in 20 l of the culture medium containing 0.1% yeastextract, 1.5% peptone, 2.0% D-glucose, 0.02% MgSO₄.7H₂O, 0.3% K₂HPO₄ and0.2% NaCl using a 30 l jar fermentor with agitation at 400 rpm andaeration of 0.5 l per minute. Cells were collected from the culture bycentrifugation at 5,000 g for 5 minutes thereafter. The weight of thepaste of cells thus obtained was 400 g.

Then, 12 g of levodione and 120 g of D-glucose were added to the cellpaste and the volume was brought to 2.4 l with ion exchanged water. ThepH was adjusted to 7.0 with 2.0% NaOH solution. The reaction mixture wastransferred into a 2 l flask and incubated at 30° C. for 15 hours withshaking at 220 rpm. After the incubation, the reaction mixture wasseparated by centrifugation at 12,000 g for 5 minutes. The volume of thereaction mixture thus obtained was 2.2 l, and the optical purity, theyield and the concentration of actinol were 96% e.e., 93% and 4.6 g/l,respectively.

EXAMPLE 7

The reaction mixture (10 l), prepared as described in Example 6, wasmixed with ethyl acetate (10 l) to extract actinol. The ethyl acetatephase (7.5 l) was separated and 350 g of active carbon powder were addedthereto for decolorizing it. After stirring for 10 minutes, the carbonpowder was removed by filtration. 600 g of anhydrous Na₂SO₄ were addedto the 6.5 l of ethyl acetate solution for dehydration. After a fewminutes of stirring, Na₂SO₄ was removed by filtration. The ethyl acetatesolution (6.0 l) was concentrated to 50 ml under reduced pressure at 30°C. 5 l of n-hexane were added to the concentrate thus obtained and themixture was stirred for five minutes, then cooled to 5° C. andmaintained at this temperature for 12 hours to crystallize actinol. Thecrystallized actinol was collected by filtration and then dried. Theweight of actinol crystals thus obtained was 32 g, and the purity, theoptical purity and the yield of actinol were 96%, 96% e.e and 70%,respectively.

EXAMPLE 8

Seed culture broth (150 ml) of Corynebacterium aquaticum AKU611 (FERMBP-6448) was inoculated into 3 l of the fermentation medium containing0.1% yeast extract, 1.5% peptone, 2.0% glucose, 0.02% MgSO₄.7H₂O, 0.3%K₂HPO₄, 0.2% NaCl and 0.3% levodione. The fermentation was carried outfor 48 hours at 30°C. using a 5 l jar fermentor with agitation at 250rpm and aeration of 1.5 l per minute. The pH of the fermentation brothwas controlled at 7.0 by NH₃ gas. After fermentation, the broth wasremoved and the cells were collected by centrifugation at 12,000 g for 5minutes. The optical purity, the yield and the concentration of actinolin the broth were 96% e.e., 71% and 2.1 g/l respectively.

What is claimed is:
 1. A process for making (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, which comprises contacting(6R)-2,2,6-trimethylcyclohexanedione with a microorganism, which isselected from the group consisting of Cellulomonas sp. AKU672 (FERMBP-6449), Corynebacterium aquaticum AKU610 (FERM BP-6447), andCorynebacterium aquaticum AKU611 (FERM BP-6448), and which is capable ofthe selective asymmetric reduction of(6R)-2,2,6-trimethylcyclohexanedione to (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, and recovering the resulting(4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone from the reactionmixture.
 2. A process for making (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, which comprises contacting(6R)-2,2,6-trimethylcyclohexanedione with a microorganism, which isCorynebacterium aquaticum AKU611 (FERM BP-6448), and which is capable ofthe selective asymmetric reduction of(6R)-2,2,6-trimethylcyclohexanedione to (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, and recovering the resulting(4R, 6R)-4-hydroxy-2,2,6-trimethylcyclohexanone from the reactionmixture.